- EU SPIDIA Project Update - Standardization and Improvement of Generic Preanalytical Tools and Procedures for In Vitro Diagnostics 5 th Annual BRN Symposium Bethesda, February 22 nd 2012 Dr. Uwe Oelmueller SPIDIA Coordinator (QIAGEN)
Agenda � SPIDIA Project History and Goals � Results & Status � New Technologies & Tools � Pan-European Guidelines � Biospecimen Quality Markers
Diagnostic Workflow From Patients to Clinical Results Patient Pre-analytical Clinical Patient Analytical Assays Sample Workflow Results Patient Sample Sample Bioanalyte Anesthesia Collection Logistics Preparation Treatment e.g. Drugs e.g. Arterial Life Style Clamp Time Time 0
It is Real Problem “Preanalytical errors still account for nearly 60%-70% of all problems occurring in laboratory diagnostics, most of them attributable to mishandling procedures during collection, handling, preparing or storing the specimens”. Lippi G. et al.. Preanalytical quality improvement: from dream to reality. Clin Chem Lab Med. 2011 Jul; 49(7):1113-26. Epub 2011 Apr 25. Preanalytics Costs of ~ 460,000 $ / year in 68 % an average German hospital Analytics caused by pre-analytical errors 13 % Frost & Sullivan 2011 on behalf of BD 19 % Postanalytics
Project Main Goals � Pan-European guidelines for preanalytics (Molecular – Blood, Tissue) � New pre-analytical tools & technologies (Blood, Plasma, Tissue, Swabs) � Sample quality markers (Blood, Tissue) � Training and dissemination
Project Facts � Program European Commission FP7-HEALTH � Consortium 7 public research organizations 8 companies 1 standards organization (CEN) � Coordinator QIAGEN GmbH � Run Time October 2008 – September 2012 (prolongation request intended) � Budget 13 Mio € (9 Mio € EC contribution) � Co-operations NCI / OBBR, CLSI, EFCC, BBMRI and other international initiatives and organizations � Web page www.spidia.eu � Newsletter
Agenda � SPIDIA Project Project History and Goals � Results & Status � New Technologies & Tools � Pan-European Guidelines � Biospecimen Quality Markers
New Tissue Fixation & Stabilization Histomorphology, IHC, RNA & DNA, Proteins Mammacarcinoma Estrogen Receptor a H&E Staining TaqMan Array Gene Signature (clone 1D5) IDC of Breast IDC of Breast 50 Formalin 45 FFPE 40 35 CRYO 30 C t FFPE Cryo 25 PFPE PFPE 20 PAXgene 15 10 5 C T N N B 1 A K T 1 I T G A V T P 5 3 A K T 2 B A X I T G B 1 T G F B 1 H P R T 1 T C F 3 P T K 2 P I K 3 R 1 T G F B R 2 T G F B R 1 F Z D 1 I T G B 3 I G F 1 I T G A 2 B K I T A X 1 8 s C O L 1 A 1 F N 1 R H O A F O S G a p d h C C N D 1 S P P 1 E R B B 2 I G F 1 R C D H 1 N F K B I A C D K N 1 B B C L 2 L 1 C D K 4 S H C 1 G S K 3 B C D K N 1 A A B L 1 A P K 1 4 S R C N F K B 1 G U S B E L K 1 V E G F A K R A S G R B 2 B C A R 1 R A C 1 B C L 2 A D 4 2 C R K N F K B 2 B R A F A P K 3 F A D D P T E N C C N D 3 J U N R E L A D V L 1 Y C C D K 2 N R A S C D K N 2 A A P K 1 A P 2 K 1 R B 1 H R A S P T K 2 B E 2 F 1 A P C C A S P 9 S O S 1 C A S P 8 B I D K D R B C L 2 L 1 1 A P K 8 R A F 1 P I K 3 C A C C N D 2 A P 3 K 5 F A S C C N E 1 F Y N L E F 1 F G F 2 E G F R C Y C S H G F C D C 4 2 C D K N 2 B N T 1 F A S L G D M M M W S M M M M M M M M genes (sorted by Ct cryo) Nucleic acid analysis superior to PFPE revealed preservation of morphology and antigenicity comparable to FFPE FFPE Kap M. et al., PLoS ONE 6(11): e27704 (2011) Viertler C. et al. , submitted for publication Groelz D. et al., unpublished data.
fcNA Profiles in Whole Blood / Plasma What is missing? � Studies for understanding fcDNA and fcRNA profile stability / changes in whole blood and in plasma � Development of fcDNA and fcRNA profile preservation technologies EDTA blood was incubated for up to 6 days at room temperature. Blood fcDNA pattern stability was determined by separating the purified plasma DNA on a 2100 Agilent Bioanalyzer Horlitz M. et al., unpublished data
Ongoing Technology & Tools Developments for Other Sample Types � Fine Needle Aspirates � Stabilization of morphology, antigenicity, DNA, RNA, proteome � Whole Blood � Stabilization of cell morphology and biomolecule profiles � Swabs � Stabilization and improved processing of respiratory and samples for molecular analysis � Stabilized Whole Blood � Integrated automated sample-to-result workflows (cellular RNA, ncRNAs incl. miRNAs)
Agenda � SPIDIA Project Project History and Goals � Results & Status � New Technologies & Tools � Pan-European Guidelines � Biospecimen Quality Markers
Evidence Based Guidelines Examples Blood DNA & RNA, Plasma fcDNA � Phase 1 Trials - Laboratories used their workflows & tools � Let by Prof. Pazzagli (Univ. Florence), supported by the EFCC � Guidelines / Standards Concepts - CEN � Phase 2 Trials - Laboratories will use SPIDIA’s optimized workflows � Guidelines / Standards Developments - CEN No. of Participants Percentage of Participants (29 who sent NA NA samples SPIDIA Trials countries) samples back sent back Blood RNA 102 93 91 % Blood DNA 130 121 93 % Plasma DNA 67 62 93 % Total 299 276 92 %
Blood DNA Trial 1 - Examples for Pre-analytical Workflow Variations � Blood storage time before DNA extraction ≤ 6 days • 39 labs: • 60 labs: 6 – 10 days ≥ 10 days • 53 labs: � Blood storage temperature before DNA extraction • 18 labs: -20 ˚C • 129 labs: +4 ˚C • 9 labs: ambient temp. � Isolated DNA storage before analysis • 20 labs: -20 ˚C • 111 labs: +4 ˚C • 27 labs: ambient temp. Pazzagli M. et al., manuscript in preparation
DNA Length Variation – Pulse Field Gel Electrophoresis 3 17 63 82 88 103 118 120 130 138 175 183 207 1 14 49 52 65 78 100 124 125 169 195 203 [kb] 194,0 145,5 97,0 195 kb 4.36 kb 48,5 23,1 9,42 6,55 4,36 � High molecular weight DNA integrity: degradation, fragmentation � High variability among samples Hartmann C. et al., unpublished results Pazzagli M. et al., manuscript in preparation
Impact of DNA quality on Immune T cell Repertoire Analysis (ImmunID Technologies) V contribution for each J gene – Research Trial (ImmunID Technologies, France) Ref. DNA from UNFI (DIV 54%) Sample 38 (Poor quality) (DIV 32%) � Lost of all long V–J rearrangements � Lost of part of intermediate length rearrangements L. Barraud et al. Unpublished data Pazzagli M. et al., manuscript in preparation
Blood RNA Ring Trial Parameters � Purity � Interference substances (RT-qPCR) � Yield � Integrity � RNA Profile Stability / Changes
Changes of Transcripts Profiles in Blood Individual Samples React Differently Human EDTA Blood stored at Room Temperature over 3 days ���� β β β β mRNA Guenther K. et al.. AMP Poster (2005)
Learning from Blood RNA Ring Trial 1 � No pooling of different donors’ blood • Accept that only sub-groups of ring trial participating laboratories get the same blood samples � No usual blood collection bags • Use dedicated EDTA bags � Immediate cooling of blood bags • Artificial gene induction and down regulation to be avoided � Use of intracellular RNA markers • External markers will behave differently
Blood RNA Second Ring Trial Preparation of Blood Samples 1 bag - 1 donor PAXgene Blood RNA Tubes Empty bag - Filled with 39 ml of EDTA solution under sterile condition - Filled with 461 ml blood BD EST Plastic Tubes from phlebotomy
Proficiency Testing for Preanalytical Workflows used for Blood RNA Analysis K. Günther, F. Malentacchi, P. Verderio, S. Pizzamiglio, C. M. Ciniselli, A. Tichopad, M. Kubista, R. Wyrich, M. Pazzagli, S. Gelmini. Implementation of a proficiency testing for the assessment of the preanalytical phase of blood samples used for RNA based analysis. Clin Chim Acta (2012) – in press.
Blood Sample Shipment - RNA Profile Changes Stabilized vs. EDTA Blood Box plots reflecting the mRNA expression of GAPDH (Panel A), IL1B (Panel B), IL8 (Panel C), and FOS (Panel D) measured in the three sample types REF, RNA A (PAXgene Blood RNA) and RNA B (EDTA). Each box indicates the 25th and 75th percentiles. The horizontal line inside the box indicates the median, and the whiskers indicate the extreme measured values. The dotted horizontal line indicates the median value of the REF samples (prior shipment) and serves for comparison. Guenther K. et al. Clin Chim Acta. 2012, in press.
Agenda � SPIDIA Project Project History and Goals � Results & Status � New Technologies & Tools � Pan-european Guidelines � Biospecimen Quality Markers
Blood RNA Quality Marker Discovery � Quality markers measuring RNA up- & down-regulation • >180 micro arrays (time course experiments) • 11 marker candidates (specific RNA degradation or gene down regulation, specific RNA gene induction, random degradation) • Technical assay validation • Next step: Performance validation within larger donor cohorts hLMNA_SFw 5.0 p=0.46 p=0.33 4.0 3.0 p=3.17*10 -6 2.0 p=4.28*10 -7 p=7.29*10 -7 1.0 0.0 0 2 6 24 48 72 -1.0 Rian E. et al. , unpublished data
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